Anthony Brown, Ph.D. - US grants

Neurofilaments are known to play a central role in the etiology of a number of human neurodegenerative disorders, most notably motor neuron disease and giant axonal neuropathy. These disorders are characterized by massive accumulations of neurofilaments in the axons of affected neurons, forming giant balloon-like swellings and leading to axonal degeneration. The accumulation of neurofilaments in these diseases is thought to be caused by changes in the mechanisms of slow axonal transport which move cytoskeletal proteins along axons. The mechanism of slow axonal transport is poorly understood and controversial. The principal issue concerns the site of assembly of cytoskeletal proteins and the form in which they move. In the case of neurofilament proteins, considerable evidence suggests that the cell body is a principal site of assembly and that these proteins are transported along the axon as assembled polymers. Alternatively, it has also been proposed that cytoskeletal proteins are transported along axons in a non- polymeric form and that the principal site of assembly is at the axon tip. To test these hypotheses, the assembly and transport of neurofilament proteins will be investigated in cultured neurons using immunofluorescence microscopy and microinjection techniques, in conjunction with quantitative digital image analysis. To identify the sites of assembly of neurofilament proteins, bovine low- molecular weight neurofilament protein (NF-L) will be microinjected into cultured rat neurons and the assembly of the injected protein will be visualized using species-specific monoclonal antibodies. In addition, the sites of assembly of newly synthesized high-molecular weight neurofilament protein (NF-H) will be identified in cultured neurons by taking advantage of its delayed expression during axon outgrowth. To visualize the axonal transport of neurofilaments, Fab fragments of neurofilament-specific antibodies will be microinjected into cultured neurons and the movement of the bound antibodies will be analyzed by immunofluorescence microscopy. To visualize the transport of neurofilament proteins regardless of the form in which they move, the accumulation of transported proteins will be investigated at an experimentally applied axonal constriction. These studies will yield important new information on the assembly and axonal transport of neurofilaments and will lay the groundwork for future studies on the mechanisms of slow axonal transport in cultured neurons, and on the mechanisms that cause neurofilaments to accumulate in axons. An understanding of these mechanisms will shed new light on the etiology of neurofilamentous neuropathies.

Neurofilaments are thought to play a central role in the etiology of a number of human neurodegenerative diseases, most notably amyotrophic lateral sclerosis. These disorders are characterized by massive accumulations of neurofilaments in the axons of affected neurons, leading to axonal degeneration. The accumulation of neurofilaments in these diseases is thought to be caused by changes in the mechanisms of slow axonal transport which move cytoskeletal and cytosolic proteins along axons from their site of synthesis in the cell body. However, these mechanisms are poorly understood and controversial. The principal issue concerns the site of assembly of cytoskeletal proteins and the form in which they move. The polymer transport hypothesis proposes that the cell body and proximal axons are principal sites of assembly of cytoskeletal proteins and the form in which they move. The polymer transport hypothesis proposes that the cell body and proximal axon are principal sites of assembly and that cytoskeletal proteins are transported in the form of moving polymers. In contrast, the cytoskeletal proteins are transported in the form of subunits of oligomers that assemble locally along the axon and the axon tip. To test these hypotheses, the assembly and axonal transport of neurofilament proteins will be investigated in cultured neurons, which are advantageous because of their accessibility to direct observation and experimentation. The proposed experiments will address two specific aims. For Specific Aim 1, immunofluorescence and immunoelectron microscopy will be combined with quantitative digital image analysis to determine the sites of assembly of biotinylated and endogenous neurofilament proteins in neurons. For Specific Aim 2, novel strategies that include constriction that includes constriction of axons will be combined with direct observation of fluorescent neurofilament proteins in living cells to determine the form in which neurofilament proteins are transported. The long-term goal of this research is to determine the mechanism by which neurofilament proteins move in axons and the mechanisms that lead to the accumulation of neurofilaments in certain neurodegenerative diseases. By testing specific hypotheses on the assembly and axonal transport of neurofilaments, the studies proposed here represent an important step toward this goal.

0089483
Doyle and Brown
With his CSIRO-Australia colleague Tony Brown, Jeff Doyle at Cornell University is continuing his highly rated studies on gene markers and genetic consequences of polyploid evolution in the perennial species of soybean, Glycine, related to the cultivated annual G. max. Polyploidy, or whole-chromosome duplication, is common in plants, and especially prevalent among the major crop plants of the world. The 17 named diploid perennial soybeans and their numerous polyploid derivatives are distributed in Australasia and the southeastern Pacific islands, and are extensively represented in the CSIRO soybean germplasm collection. One major goal is to explore in wild species, with genomic tools, ideas about gene silencing and nucleolar dominance formulated from studies on wheat and cabbage polyploids, and to extend such studies to several single- or low-copy nuclear genes, using species complexes where the parent diploids have been identified (and parent tetraploids for hexaploid and octoploid derivatives) and where multiple origins of the "same" polyploid can be documented with molecular markers from both nuclear and chloroplast genomes. Studies of the fate of duplicated (homoeologous) loci will be pursued through cloning of the PCR products, but the investigators are also exploring use of denaturing chromatographic methods to screen numerous polyploid samples for the presence of novel alleles (heteroduplexes from annealing of alleles that differ in length or sequence separate, producing diagnostic elution profiles). Preliminary data are in hand for a nuclear histone gene (H3-D), for nuclear glutamine synthetase, for cox2 (transposed from the mitochondrion in many legumes), and the ribosomal RNA genes. In collaboration with Julie Vogel at DuPont, with access to DuPont's 200,000 soybean cDNA clones, the investigators also plan to screen for candidate nuclear genes showing silencing (through differential hybridization with diploid and polyploid DNAs), and concentrate on those that function in the chloroplast or mitochondrion (and hence where the paternal homoeologue is thought likely to be silenced).

[unreadable] DESCRIPTION (provided by applicant): Cytoplasmic accumulations of neurofilaments are a hallmark pathological feature of a number of human neurodegenerative diseases, most notably amyotrophic lateral sclerosis. These neurofilamentous accumulations are thought to be caused by changes in the mechanisms of slow axonal transport, which move cytoskeletal and cytosolic proteins along axons from their site of synthesis in the nerve cell body. We have recently observed the slow axonal transport of neurofilament protein in cultured nerve cells. The proteins move in the form of filamentous structures that may represent single neurofilament polymers. Contrary to the widely held view that slow axonal transport is a slow, synchronous and exclusively anterograde movement, we found that the filaments actually move at very fast rates, approaching the rate of fast axonal transport, and that the movements are also infrequent, bi-directional and highly asynchronous. Based on these observations, we have proposed a new model for slow axonal transport in which the actual rate of movement is fast, but the overall rate is slow because the rapid movements are interrupted by prolonged pauses. In this application, we propose to use live-cell fluorescence imaging strategies to test specific aspects of this hypothesis. In Aim 1 we will test the hypothesis that the moving filaments represent single neurofilament polymers. We expect that these experiments will also reveal the tracks along which the filaments move. In Aim 2 we will test the hypothesis that moving and stationary filaments differ in their phosphorylation state at specific epitopes and that they differ in their association with specific microtubule motor proteins. In Aim 3 we will test the hypothesis that rapidly moving filaments are delivered to the tip of growing axons in sufficient quantity to support the elaboration of the axonal neurofilament array during axon growth. We will also test the hypothesis that the growth cone is a site of frequent reversals in the direction of filament movement and that the frequency and/or directionality of filament movements in the distal axon is regulated in response to the rate of axon growth. The long-term goal of our research is to determine the mechanism and regulation of neurofilament protein transport along axons and the mechanisms that lead to the accumulation of neurofilaments in neurofilamentous neuropathies. [unreadable] [unreadable]

confocal scanning microscopy; nervous system disorder; biomedical facility; model design /development; time resolved data; biological models; lasers;

Despite the fact that the legume genus Glycine includes the cultivated soybean (G. max), much remains to be learned about its evolutionary history and taxonomic relationships. These include the origin of the entire genus associated with an ancient genome duplication event, relationships among the reproductively isolated "genome groups" to which the approximately 25 diploid perennial species of the genus belong, and the complex history of genetic differentiation among closely related species within these genome groups. All of these issues have in common the possibility that hybridization has played a role in producing the patterns observed in DNA variation from nuclear, chloroplast, and mitochondrial genes. Polyploidy (multiple sets of chromosomes) and hybridization separately, or together in allopolyploidy, are two of the most important processes that shape the genomes of plants, including cultivated plants such as wheat, maize, cotton, and soybean. In the case of Glycine it is unknown whether hybridization was involved in polyploid formation, as hypothesized to have occurred 15 million years ago, and if so, what species were the donors of the two genomes. This and other questions at progressively more recent stages in the history of Glycine that involve the role of polyploidy and hybridization can be addressed by studying a large number of genes on the different chromosomes or in different genomic compartments (mitochondrion, chloroplast). The largest source of such genes is the nuclear genome, and this project taps genomic projects in soybean and other legumes (e.g., Medicago truncatula) to provide many candidate genes for study. Around 100 nuclear genes will be assessed for their utility in addressing phylogenetic questions, taking into account such additional criteria as position on the soybean genetic map. Analytical methods will include phylogeny reconstruction using various approaches, assessment of incongruence, testing for polytomies and hybridity, network construction, and admixture assessment. The work will address general issues of dealing with incongruence, identifying and using low copy nuclear genes, and studying polyploidy.
Achieving a better understanding of polyploidy and hybridization in general, and particularly in Glycine, is of broad relevance and significance for plant biology and genetics because of the prevalence of these phenomena. The project will have an impact on the infrastructure of science by training undergraduate and graduate students and a postdoctoral fellow, including minority undergraduate students who will participate in summer research internships. Collaboration with the Cornell Institute for Biology Teachers will lead to the development of phylogeny laboratories for high school and middle school students. The project is international in its scope, with researchers from the United States and Australia, and involves the use and enhancement of germplasm resources in the form of Glycine seed collections in both countries.

Nerve cells communicate by conducting electrical signals along slender cytoplasmic extensions known as axons. Animals have evolved two basic mechanisms for increasing axonal conduction velocity. One is to increase axonal diameter and the other is to insulate axons by a process called myelination, which is a tight spiral wrapping of the axons that is formed by myelinating cells. In vertebrates the growth of axon diameter is caused principally by the accumulation of space-filling cytoskeletal polymers called neurofilaments inside the axons, and this is regulated locally by chemical signals from the myelinating cells. It is known that neurofilaments are transported along axons and that they alternate between rapid movements and prolonged pauses. The proportion of the time that the neurofilaments spend pausing is likely to be a principal determinant of their residence time in axons. This is a collaborative experimental and modeling project involving a biologist at Ohio State University and a physicist at Ohio University. The central hypothesis to be tested is that myelinating cells control axonal caliber by regulating neurofilament pausing. A computational model will be developed that relates the moving and pausing behavior of neurofilaments to their distribution along axons. The model will be based on detailed kinetic parameters of neurofilament movement derived experimentally in cultured neurons and will be verified experimentally by fluorescence microscopy of neurofilament movement in myelinated axons in tissue culture. The proposed research will generate a rigorous and quantitative framework that relates the size and shape of axons, which is a key influence on their electrical properties, to the moving and pausing behavior of their internal constituents. The research will involve graduate and undergraduate students in both the physical and biological sciences, providing an integrated and cross-disciplinary training experience at the interface between computational and experimental biology.

Fluorescent-Magnetic Nanomanipulators for Cytoskeletal Mechanical Investigations

Abstract

The research objective of this award is to develop a new methodology to examine the role of mechanical force in cell migration. The approach consists of fluorescent-magnetic nanomanipulators that impart force to the cell?s internal scaffolding (i.e., the cytoskeleton) through stimulation by custom-designed magnetic instrumentation. Cell response is observed via the nanomanipulators? fluorescence property. This research will focus on examining migration of cells crucial to the wound healing process (i.e., fibroblasts). Fluorescent-magnetic nanomanipulators will be characterized and optimized to interact with magnetic instrumentation; proof of concept of nanomanipulator delivery to the cell interior and manipulation potential will be demonstrated; and the effect of force applied by nanomanipulators on cell migration will be evaluated in fibroblasts. Deliverables include optimized nanomanipulators and magnetic instrumentation, validation of the intracellular force application method, documentation of research results in the fibroblast model, engineering student education at the graduate and undergraduate level, and a module based on this research for public school outreach disseminated by COSI, a local science museum.

If successful, this research will provide new tools to explore the influence of mechanical force on protein expression and cellular processes, in particular those that underlie cell migration. Cell migration is a critical component of several biological events including wound healing, fetal development, and cancer metastasis. This research would provide methods to apply force to subcellular components with the smallest manipulators yet studied (~ 10-50 nm), sizes that are similar to many of the components of interest (e.g., protein receptor, cytoskeletal element). Research results will be disseminated through journal publications, conference presentations, and also, to the general public, through interactions with an area science museum. Graduate and undergraduate students will participate in the research, and results will be disseminated through classroom materials as well.

PROJECT SUMMARY/ABSTRACT Neurofilaments are space-filling cytoskeletal polymers that function to increase the cross-sectional area of axons. These polymers are transported along axons at fast rates, but the overall rate is slow because the movements are interrupted by prolonged pauses. Neurofilaments accumulate in axons during the growth of axonal caliber and they also accumulate abnormally and excessively in a wide range of neurodegenerative diseases, most notably amyotrophic lateral sclerosis. Mutations that disrupt neurofilament assembly also cause one form of Charcot-Marie-Tooth disease. The long-term goal of our research is to understand the transport and assembly dynamics of neurofilaments in axons and the mechanisms that cause neurofilaments to accumulate in development and disease. We propose three specific aims. Aim 1 is to investigate the assembly dynamics of neurofilaments in axons. Axonal neurofilaments can be 100 ¿m or more in length in vivo and they can also exchange subunits with a soluble pool, but little is known about how these processes occur. We propose that neurofilaments can lengthen by end-to-end annealing of pre-formed filaments and that they can exchange subunits by addition and loss of subunits along the length of the filaments, a process that we term intercalary subunit exchange. We will use cell fusion as well as photobleaching and photoactivation strategies to test these hypotheses in cultured neurons. We will also extend our studies to other types of intermediate filament proteins to establish whether our findings are more generally applicable. Aim 2 is to investigate the role of phosphorylation in neurofilament transport. We propose that phosphorylation of neurofilaments by CDK5 and ERK1/2 kinases regulates their transport in an additive and dose-dependent manner by increasing the proportion of the time the neurofilaments spend pausing. We will test these hypotheses in cultured neurons by using site-directed mutagenesis to mimic phosphorylated or non- phosphorylated states at specific sites. We will also manipulate kinase activities directly using pharmacological inhibitors and constitutively active or dominant negative kinase constructs. Aim 3 is to investigate the functional significance of neurofilament pausing. We propose that neurofilament pausing is a critical determinant of axonal neurofilament content. We will test the hypothesis that neurofilament phosphorylation causes axonal neurofilaments to accumulate by increasing their residence time in axons. We will also use long-term myelinating cultures to test the hypothesis that myelinating cells locally slow neurofilament transport by increasing the proportion of the time that they spend pausing. The use of live-cell imaging to study neurofilament transport in myelinating axons in culture is a particularly innovative aspect of this Aim. The mechanisms that regulate neurofilament pausing are likely targets for disease processes that lead to excessive accumulation of neurofilaments in axons.

CORE E: IMAGING CORE Overview This Core provides two services 1) A Confocal Microscopy established during the prior funding period, and 2) A Magnetic Resonance Imaging (MRI) which is a new addition. The Imaging Core has been a highly successful workhorse facility during the current P30 award, serving neuroscience Investigators in 17 different laboratories during the past 5 years leading to 13 papers and two new NIH grants. The MRI core will leverage considerable Institutional investment in MRI at OSU, including the formation of a new state-of-the-art Small Animal Imaging Center, and addresses a pressing need for technical support with the use of this powerful Imaging technology.

Nerve cells communicate by sending electrical impulses along thin protrusions called axons. One mechanism by which animals increase velocity of electrical impulses is to expand axons' cross-sectional area. This expansion is caused by an accumulation of microscopic space-filling protein polymers called neurofilaments, which are transported into axons where they form a dynamic scaffold. This collaborative project will use microscopic imaging in conjunction with computational modeling to test the hypothesis that neurofilament accumulation in axons is caused by a slowing of neurofilament transport, much as cars pile up on highways when the traffic slows. This project will provide a rigorous and quantitative framework that relates the size and shape of axons, which is a key influence on their electrical properties, to the motile behavior of their internal constituents. This work will also shed light on the mechanism by which neurofilaments accumulate abnormally and excessively within axons in many neurodegenerative diseases.

This project will provide a unique training opportunity at the interface of computational and experimental biology for students with diverse backgrounds in the physical and life sciences. An emphasis will be placed on mentoring undergraduates lacking prior research experience, including women and minorities, through annual Research Experiences for Undergraduates fellowships. The investigators will also reach out to undergraduates by participating together in the Research for Undergraduates: Adventures in Mathematical Biology and its Applications curriculum at Ohio State University, which is an NSF-funded Undergraduate Biology and Mathematics program, and by serving as joint mentors in the Undergraduate Summer Research Program of the NSF-funded Mathematical Biosciences Institute at Ohio State University. Lastly, a neurofilament wiki page will be developed featuring information and resources on mathematical modeling of axonal transport to support these outreach activities and to facilitate the exchange of data between the two laboratories.

DESCRIPTION (provided by applicant): We are requesting funds to purchase a PerkinElmer UltraVIEW spinning disk confocal microscope for live imaging of cultured cells, tissues and embryos. The microscope will be housed in the Imaging Core Facility of the Center for Molecular Neurobiology on the West Campus of Ohio State University. Established about 25 years ago, the Center is a dynamic and interactive group of 13 faculty who have joint appointments in various Colleges. The Center has its own Director and support staff and excellent space and resources, including an adjacent mouse facility and a zebrafish core facility. The spinning disk confocal microscope purchased with this award represents critical and enabling technology that will support the research of 7 Center faculty (4 major users and 3 minor users) who are studying problems in neuroscience and cancer, and who collectively hold 9 active NIH grants totaling more than $2.4 million dollars in annual NIH funding. The Imaging Core Facility was established 6 years ago by Dr. Anthony Brown (the PI of this proposal) with funds from an NIH P30 Institutional Center Core Grant. The facility consists of a Leica TCS SL single-point scanning confocal microscope and a Zeiss Axiophot epifluorescence microscope with camera and software. Dr. Brown is the Director of the facility and supervises the Manager of the facility, Ms. Paula Monsma. Ms. Monsma manages al day-to-day operations, including training, user support, scheduling and equipment maintenance. Dr. Brown is a Professor in the Center for Molecular Neurobiology and an expert in live-cell imaging with more than 25 years of experience using light and electron microscopy in his research. Dr. Brown maintains an active and productive research program involving live-cell imaging of axonal transport and the cytoskeleton of nerve cells and has been funded by the NIH continuously for the past 15 years. Ms. Monsma has a Masters degree in cell biology and 15 years experience as a laboratory research assistant, including 5 years running a DNA sequencing core facility and 6 years running the Imaging Core Facility in the Center for Molecular Neurobiology. The P30 Center Core grant, which was recently renewed for another 5 years, provides 45% salary support for Ms. Monsma, enabling her to devote 2-3 days a week to her Imaging Core facility duties. In summary, this proposal leverages the expertise and resources established by our P30 Institutional Center Core Grant to acquire a critical and enabling new technology that will support the live-cell confocal imaging needs of 9 NIH-funded research projects in neuroscience and cancer. PUBLIC HEALTH RELEVANCE: Modern biomedical research is now critically dependent on direct observations of fluorescent molecules in living cells, tissues and organisms. This grant wil fund the purchase of an advanced state-of-the-art confocal fluorescence microscope for this purpose. The shared instrument will form part of the Imaging Core Facility in the Center for Molecular Neurobiology at Ohio State University, and it will provide vital new technological capabilities to support the research of 9 NIH-funded research projects in neuroscience and cancer.

CORE A (ADMINISTRATIVE) ABSTRACT The Administrative Core provides fiscal and operational oversight of the Center, sets policies, manages the budget, and provides administrative support to the PI and Core Directors for these matters. The PI chairs the Steering Committee, consults with the External Advisers, and works with the Core Directors to run the Scientific Cores in an efficient, responsive and transparent manner that addresses needs of NINDS-funded and other neuroscience investigators across campus. The Core communicates core services and activities to neuroscience users, tracks personnel, expenditures, scheduling, usage and productivity, generates billing and progress reports, and convenes Neuroscience User Group meetings to promote core services and stimulate new cross-core collaborations.

CORE E (IMAGING) ABSTRACT The Imaging Core provides access to well-maintained point-scanning and spinning disk confocal microscopes as well as expert consultation, training and assistance with confocal fluorescence microscopy of cells, tissue slices and zebrafish embryos, both in the living and fixed state. Advanced capabilities include long-term 4D multi-color time-lapse imaging, simultaneous dual-color imaging, fluorescence photoactivation and FRAP, FRET, image stitching and 3D-rendering. The Core can perform imaging sessions with investigators, if necessary, and can provide training and advice on image processing and analysis.

The Ohio State University (OSU) Neuroscience Center Core, now in its 12th year of NINDS support, provides specialized expertise and services that support research into the causes and treatments of neurological disorders. The Center serves a total of more than 40 neuroscientists (20 NINDS-funded) from multiple departments, centers, and institutes, and has become a catalyst for neuroscience research and collaboration across campus. Areas of strength include basic and translational research on neurodegenerative and neuromuscular diseases, brain tumors and neurotrauma. The Center leverages substantial institutional investment and federal support in research infrastructure and neuroscience at OSU. This includes the recently formed Brain and Spine Hospital and the Neuroscience Research Institute, a multi-million dollar effort that aligns the activities of about 175 basic and clinical neuroscientists across campus. The Center comprises one Administrative Core and four Scientific Cores, all of which are established, functional and successful. The Scientific Cores provide access to services, equipment and expertise not otherwise available to individual PIs, enhancing their ability to execute the aims of their funded projects and facilitating their adoption of new approaches and technologies. This centralization of expertise and equipment in the Scientific Cores increases the efficiency and quality of NINDS-funded research at OSU by minimizing duplication of effort and equipment, and ensuring the uniform application of best practices. The Cores are directed by investigators with deep and proven expertise in the Core services. Core A (Administrative) sets policies, oversees the Core operations and budget, and facilitates communication of Core services to neuroscientists on campus. The Core arranges meetings of the Neuroscience User Group in order to promote core services, identify new needs, and promote cross-core collaborations. Core B (Injury) provides equipment, training and technical expertise, including standardized and well-characterized injury protocols, to support research on spinal cord and brain injury models in rodents. Core C (Behavior) provides access to the equipment and skilled technical expertise necessary to perform comprehensive behavioral phenotyping of rodent models, as well as expert training and consultation on the execution of behavioral experiments. Core D (Electrophysiology) provides specialized equipment, training and technical expertise necessary to monitor and record the electrical activity of neurons and glia. Core E (Imaging) provides access to confocal microscopes including expert training, consultation and assistance with fluorescence imaging of living and fixed cells, tissues and embryos. Collectively these Cores will support 23 NINDS-funded projects (including 12 qualifying R01 projects) totaling $7.5 million dollars in annual funding, plus 14 other NIH-funded neuroscience projects totaling $3.9 million in annual funding, enhancing the research environment for these projects and fostering a cooperative and interactive research environment in which multi-disciplinary approaches and joint research efforts are stimulated.

Nerve cells extend long, thin protrusions called axons that define the wiring pattern of the nervous system. Axons allow nerve cells to communicate electrically with each other and with other cells throughout the body. Each axon contains a microscopic, internal scaffold of space-filling proteins called neurofilaments that are constantly shuttled along the axon by molecular motor proteins; these define axon shape and size. Neurofilaments accumulate during development, increasing axon diameter and allowing electrical activity to travel more quickly; excessive accumulation (as occurs in many neurodegenerative diseases) can lead to communication abnormalities and axonal degeneration. This project tests the hypothesis that the rate of neurofilament transport determines the diameter, shape and function of axons. The work will be conducted by a seasoned interdisciplinary team of biologists and physicists, combining innovative biological imaging techniques with mathematical and computational methods to investigate these important questions. The insights gained from this research will be critical for understanding healthy brain function and could also provide important insights into the axonal problems observed in many neurodegenerative diseases. Trainees on this project from both the physical and life sciences will work in teams supervised by the principal investigators, and will expand their skills through interdisciplinary interaction, adding to the skilled research workforce at the interface of the physical and life sciences. To extend the impact of the proposed research to the K-12 level, the physicists and biologists on this project will host focused, small-group workshops that will seek to empower middle and high school teachers with ideas and tools to invigorate their instruction in the areas of cell biology and algorithmic thinking, and introducing freely available but powerful learning tools that they can apply in their classrooms.

The function of nervous systems is dependent on the propagation of action potentials along axons at a velocity that is specific to their physiological function. This velocity is dependent on axon size and shape. A principal determinant of axon size and shape in vertebrates are space-filling cytoskeletal polymers called neurofilaments. Neurofilaments are also cargoes of axonal transport that move along microtubule tracks. Thus, neurofilaments define axonal morphology, but they are also in constant flux. The proposed research addresses this intriguing and physiologically important relationship. The central hypothesis is that the kinetics of neurofilament transport determines axonal neurofilament content, which in turn specifies axonal caliber and function. The specific goals are to determine the dynamic interplay between neurofilament transport velocity and flux in the specification of overall axon caliber, and how neurofilaments navigate local constrictions at the nodes of Ranvier. To accomplish these goals, the investigators will employ a tight integration of computational and mathematical methods with innovative live imaging of myelinated axons in peripheral nerves ex vivo from a new transgenic mouse that expresses a photoactivatable neurofilament protein in neurons.

PROJECT SUMMARY/ABSTRACT We are requesting funds to purchase a Zeiss LSM 800 point-scanning confocal microscope with Airyscan detector to support NIH-funded research on the West Campus of Ohio State University. This instrument represents essential and enabling technology that will support the research of 13 PIs (8 major users and 5 minor users) with a particular strength and focus in neuroscience and neuromuscular research. These users hold 23 active grants totaling $7.1 million in annual funding, including 16 NIH research grants totaling $5.6 million and 7 research grants from NSF and private foundations totaling $1.5 million. All 8 of the major users and 3 of the minor users are NIH-funded. NIH-funded users and NIH-funded major users will account for 80% and 70% of the Available Usage Time, respectively. The instrument will replace an obsolete Leica TCS SL point-scanning confocal microscope and will provide a modern and reliable instrument offering substantial and much-needed improvements in sensitivity for imaging of fixed cells, tissues and zebrafish embryos, as well as additional laser lines, higher scan resolution, a rotatable scan area, and a motorized stage. Together, these will enable multi- wavelength imaging, image cropping and tiling capabilities not available on the current instrument. The Airyscan detector will offer greatly improved detection and image quality due to impressive additional gains in sensitivity, as well as a 1.7-fold improvement in spatial resolution. The instrument was selected after extensive research and consultation as well as on-site testing of two systems and will be housed in the Neuroscience Imaging Core, which is an established and successful core facility of the Ohio State University Neuroscience Center Core that is supported by an NIH P30 Center Core grant (Brown, PI). Importantly, this is the only confocal imaging core facility within 1.4 miles of the West Campus of Ohio State University and is thus critical for NIH-funded research on this part of campus. Established in 2004 and now in its 14th year of NIH funding, the core facility is directed by Dr. Brown who is a cell biologist with more than 25 years of experience in fluorescence microscopy. Dr. Brown supervises the Facility Manager, Ms. Monsma, who manages all day-to-day operations, including training, user support, scheduling and equipment maintenance. Ms. Monsma has an MS in cell biology and extensive experience in confocal microscopy including first-author publications and 13 years of experience managing this facility. The NIH P30 Center Core grant was recently funded for another 4 years and provides salary support for Ms. Monsma, as well as the confocal service contract. An established user fee structure, billing mechanism and financial backing from the College of Medicine guarantee continued sustainable operation when the P30 ends. Thus, this proposal leverages substantial institutional support, established expertise and resources to acquire a modern instrument that will support the aims of a large number of NIH-funded researchers and projects.